Pertussis toxin (PTX) is a hexameric protein produced by Bordetella pertussis and is comprised of five different subunits termed S1 through S5. PTX exerts a diverse array of biological effects in vitro and in vivo, and is believed to occupy a central role in the pathogenesis of whooping cough or pertussis. Many of these effects appear to result from the ability of PTX to catalyze the ADP-ribosylation of guanine nucleotide-binding proteins of the adenylate cyclase complex, using NAD+ as the ADP-ribose donor. This enzymatic modification results in impaired regulation of cyclic AMP production with consequent perturbation of cell function. The ADP-ribosyltransferase activity is associated with the S1 subunit (Mr 28,000) of PTX. PTX has also been shown to be a major antigen in acellular vaccines. One of the goals of our laboratory is to study the structure-function relationships of this protein. In the light of the current discussions about the safety of pertussis vaccines, a better knowledge of the enzymatic mechanism of PTX is of particular interest because it could lead to the development of an enzymatically inactive but immunologically intact toxin, able to elicit protection against the disease without the harmful side effects. Our most important scientific advance derived from the gene expression experiments was the discovery of a molecular approach for inactivation of pertussis toxin biological activity. Using site-specific DNA mutagenesis, the S1 subunit was modified by a single amino acid substitution. This mutation virtually eliminated enzymatic activity, yet the immunogenic protective epitope was retained. In order to take advantage of this important finding, we have devised several methods to transfer these genetic changes into the chromosome of B. pertussis. Thus, a genetically detoxified pertussis toxin molecule has been produced using the new mutant strain of bacteria. This genetically modified holotoxin should have the same immunogenic properties as native pertussis toxin and it can be used as a vaccine antigen without chemical inactivation.